Various scientific and scholarly articles are referred to in parentheses throughout the specification, with full citations appearing at the end of the specification. These articles are incorporated by reference herein to describe the state of the art to which this invention pertains.
The major seed storage proteins of maize are referred to as zeins. In normal maize genotypes, zeins constitute 50-60% of the total endosperm protein at maturity. Zeins are a heterologous group of proteins that can be classified by sequence homology and size (reviewed in Ueda, T. and Messing, J., 1993). The α zeins are the largest subgroup (zein-1), encoded by about 65 genes, and soluble in ethanol under nonreducing conditions. Most of these have a molecular weight of 19 kDa, with one subfamily having a molecular weight of 22 kDa. The other subgroup (zein-2) consists of the β, γ and δ zeins that are soluble in ethanol under reducing conditions. They differ in amino acid composition and sequence homologies.
One δ zein gene encoding a 15 kDa zein has been cloned. This zein was found to encode a protein with a moderate level of methionine (11%). Two cloned γ zeins of 16 and 27 kDa molecular weight were found to be very high in proline. Two δ zeins have been cloned, encoding 10- and 18-kDa proteins rich in methionine (22%, Anderson Kirihara et al., 1988, and 28%, Swarup et al., 1995).
As determined from genetic studies, zeins are regulated at the transcriptional and post-transcriptional level. Differences in regulation occur in a subfamily-specific manner. For instance, opaque-2 (o2) variants prevent the transcriptional activation of 22-kDa α zein genes. Also, as described in more detail below, the dzr1 locus regulates the accumulation of 10-kDa δzein mRNA (Cruz-Alvarez et al., 1991; Schickler et al., 1993).
Like many cereal storage proteins, zeins are deficient in lysine, tryptophan and methionine. For this reason, corn meals used in animal feeds (particularly for monogastric livestock such as poultry) are supplemented with legume (mainly soy) meals to increase the levels of lysine. However, the corn-legume mixture is still deficient in methionine, and processed methionine is often added as a supplement to this mixture. Similarly, it is likely that the methionine level is limited in cereal-legume mixtures, comprising human diets in many third-world communities. Supplementation of cereal-legume mixtures with processed methionine is costly (estimated at about one billion dollars for the U.S. feed business), and in many instances, infeasible. Improving the amino acid composition of maize and other cereals to include more lysine, tryptophan and methionine is therefore an important agronomic objective. Even modest increases in one or more of these amino acids, particularly methionine, in maize and other cereals could lead to a reduced need for processed methionine supplements or added soybean meal.
One approach to producing animal feed with an increased methionine content is to genetically engineer the feed plant (e.g., maize or soybeans) to produce or retain more methionine. Since composition and differential accumulation of various storage proteins, rather than amino acid biosynthesis, is the limiting step, it is the seed proteins themselves that must be engineered, either for altered composition or for enhanced expression.
Using natural variation and crosses of variant inbred lines, Messing and Fisher (1991) produced a maize hybrid, BSSS53xMo17 having a five-fold enrichment of methionine in the prolamin fraction, as compared to the reciprocal hybrid Mo17xBSSS53 with Mo17 as the female parent, sufficient to replace the processed methionine supplement in a soybean-corn diet. When such an enriched diet was tested in a two-week feeding trial of one-day-old chicks, the high methionine maize was demonstrated as a nutritious protein source. The increase in methionine in these hybrid seeds was the result of increased expression of the 10-kDa δ zein gene.
In spite of the successful production of the high methionine BSSS53xMo17 maize hybrid, most hybrids, like the reciprocal cross of Mo17xBSSS53, exhibit an inhibition of the overexpression of the 10-kDa zein gene, by a heretofore unknown mechanism, thereby preventing the use of the high methionine (HM) phenotype for animal feed. Genetic analysis has revealed three single loci related to the HM phenotype (Benner et al, 1989; Swarup et al., 1995): (1) the 10-kDa δ zein locus (dzs 10, delta zein structural gene 10) on the long arm of chromosome 9, the 18-kDa δ zein locus (dzs 18) on the long arm of chromosome 6, and the dzr1 (delta zein regulatory gene 1) formerly called Zpr10/22, on the short arm of chromosome 4. Certain alleles of dzr1 provided the first example in which zein gene expression is controlled by parental imprinting (Chaudhuri and Messing, 1994).
Transcriptional run-on experiments indicate that the lower level of expression of the 10-kDa zein gene in Mo17, as compared to BSSS53, is due to mRNA accumulation rather than transcription (Schickler et al., 1993). This differential expression was found to be due to different alleles of drz1 (Chaudhuri and Messing, 1994). Moreover, heteroallelic combinations of these two alleles result in reduced 10-kDa mRNA levels, indicating that the drz1+Mo17 allele is a negative dominant allele. The result of the presence of this negative dominant allele in Mo17 or any other inbred variety bearing the allele is that hybrids generated therefrom will have reduced expression of the 10-kDa zein gene, even if the other parent overexpresses the gene, either naturally or by genetic engineering.
As an example, U.S. Pat. No. 5,508,468 to Lundquist et al. discloses a fertile hybrid transgenic maize plant regenerated from immature embryos of a cross between A188 and B73, transformed with a chimeric 10-kDa zein gene controlled by the promoter for a 27-kDa zein gene. If such a plant is crossed with Mo17 or any other variety carrying the dominant negative allele of dzr1, any overexpression of the 10-kDa zein transgene (or native gene) that might be seen in the parent will be reduced or lost in the progeny, due to the presence of the dominant negative dzr1 allele.
Clearly, the presence of the negative dominant dzr1 allele is detrimental to the use of a 10-kDa zein gene for increasing methionine content in maize or any other plant. It would be of agronomic and economic significance, then, to identify the mechanism(s) by which the negative allele functions, and to devise methods and biological molecules to circumvent or alleviate such function. On the other hand, circumstances can be envisioned by which the negative function is desirable. Certain gene products that are highly expressed throughout plant development should be specifically reduced during seed maturation and therefore prevented from entering the food chain. Such an example might be the Bacillus thuringiensis insecticidal protein, which is needed for insect damage protection but not in the seed flour.